This proposal is designed to elucidate the molecular mechanisms underlying voltage-dependent gating of the sodium (Na) channels from adult human skeletal (hSkM1) or cardiac muscle (hH1), expressed heterologously either in a mammalian cell line or in Xenopus oocytes. The experiments will entail primarily electrophysiological recordings (single channels, macroscopic ionic currents, and gating currents) of wild-type and mutant channels, but will also include fluorescence measurements of single voltage-clamped cells. The first project is to study the relationship between the movement of S4 transmembrane segments and the voltage-dependent gating of hSkM1 channels. Initial studies will concentrate on the S4 segment of domain 4 (i.e. S4/D4). Cysteine replacements of each of the 8 basic residues of S4/D4 will be used to determine the accessibility, both extracellular and intracellular, of specific residues to cysteine reagents during the voltage-dependent movements of this transmembrane segment. The voltage dependence and kinetics of S4 movement will be determined from the reaction rates of these reagents. We will also obtain a lower-bound estimate for the distance between the extracellular mouth of the ion- conducting pore and the extracellular end of S4 segments, by testing whether extracellular pore blockers prevent access of 54 residues to chemical reagents. Later studies will include experiments on S4 segments in other domains and measurements of voltage-dependent changes in fluorescence intensity for fluorescently labelled S4 residues. The second project is to test the hypothesis of a physical connection between a putative cytoplasmic inactivation gate and an activation voltage sensor of the hH1 Na channel. We will also test the possibility that the cytoplasmic linker between S4 and S5 segments in D4 acts as a receptor for the inactivation gate.